In general, structured illumination light may be understood as any light with a spatially variable distribution of intensity over the cross-section of the light beam. In particular, light with a periodic distribution of intensity over the cross-section can be used, for example, a linear patterns with illuminated and not-illuminated lines.
Such a pattern can be generated by a linear grating on the sample plane.
Various methods are known to provide different grating orientations. In principle, a single grating may be used and rotated into different orientations. In addition to a grating, optic image field rotators may also be used, for example an Abbe-König prism. This way, a rotation of the image field and thus a rotation of the cross-sectional area of a light beam can be generated. Furthermore, several differently aligned gratings may be provided, with it being possible to select respectively one of them by a motorized grating exchanger. Additionally, an optic beam splitting can occur to generate interfering radiation or superimposed, differently aligned gratings may be provided on a substrate and means for selecting a desired diffraction order.
In a generic optical arrangement, an optic carrier is provided for positioning in a radiation path of a light microscope, at which a first set of optical assemblies is arranged to generate structured illuminating light with different orientations. These optical assemblies generally represent gratings of various orientations. The optic carrier can therefore also be called a grating carrier.
In the prior art, gratings at an optic carrier are exchanged by rotating the optic carrier. This way, only one of the present gratings in the radiation is present in the radiation path of the light microscope at any given time. Here it is disadvantageous that an exchange between different grating orientations is relatively time consuming. This period, also called dead time, amounts typically to several hundred milliseconds in conventional optical arrangements and light microscopes.
In other optical arrangements of the prior art, the dead time for exchanging two grating orientations is relatively long, too. Additionally, conventional designs are frequently expensive with regards to mechanics, perhaps requiring high precision of positioning of mobile optic components, and also lead to undesired loss of intensity of the light beam.
An objective of the invention is providing an optical arrangement and a light microscope, by which in a simple design a rapid exchange is possible between various grating orientations in order to generate structured illumination light.
This objective is attained in the optical arrangement with the features of claim 1 and a light microscope with the features of claim 19.
Preferred embodiment variants of the optical arrangement according to the invention and the light microscope according to the invention are the objective of the dependent claims and are explained in the following description, particularly in the context with the figures.
An optical arrangement of the type mentioned at the outset includes an adjustable deflection device for a selectable deflection of a light beam to one of the optical assemblies and for deflecting a light beam, coming from these optical assemblies, in the direction of a sample to be examined.
The light microscope of the type mentioned at the outside has an optical arrangement according to the invention in a radiation path between the light source and the sample plane.
A fundamental idea of the invention is the fact that no motion of the grating itself, or even the optic carrier with the gratings, is necessary for inserting a certain grating into the radiation path of the light microscope. Due to the relatively large dimensions and weight of the gratings as well as the optic carrier, here any movement thereof is time consuming. This problem is aggravated in that the gratings must be positioned with high precision in order to adjust the grating phase shifts per grating orientation necessary for this microscopy method. According to the invention, in order to select a grating image, with a certain orientation, the movement of the grating can be omitted by selectively adjusting the radiation path of a light beam for illuminating the sample to one of the optical assembles at the optic carrier. The optical assemblies preferably comprise gratings, which are arranged in various orientations in reference to each other. Any mechanical movement is here only required in the direction of deflection, if at all.
The movements at the deflection device are considerably smaller than any movement of a grating into the radiation path or out of it, so that a grating selection can occur via the deflection device in a particularly short period of time.
Another core concept of the invention is that with the adjustable deflection device, the radiation path of a light beam coming from one of the optical assemblies can be influenced in a variable fashion. This way a radiation path of the light beam from the deflection device in the direction towards the sample to be examined can be independent of the selection of the optical assembly.
Due to the fact that the optic carrier as well as the optical assemblies fastened thereat can be embodied in a spatially fixed fashion, advantageously a relatively large number of optical assemblies is possible at the optic carrier without this negatively influencing the time frame for selecting one of the optical assemblies. This way, the first set of gratings with identical grating constants may show at least three, or also at least five or at least seven gratings. This way, high precision of measuring can occur with yet low dead times between any exchange of the gratings selected.
In light microscopes of the prior art, the necessary time for image recording is determined by a combination of image field rotators and gratings using the following parameters: rotations of the image field rotators to generate structured illuminating light of various orientations show an adjustment period of approximately 300 ms. At least 3 rotation settings must be approached. Additionally, images must be recorded for 3 phases of each orientation of the image field rotator and grating. Thus, 9 images must be recorded. The exposure time for recording an image of the sample amounts to 30 ms, for example. Accordingly, the overall measuring period comprises in prior art approximately 3*300 ms (rotation of the grating image)+9*30 ms (exposure time)+9*10 ms (phase shift over Piezo element), thus more than 1.2 seconds. Contrary thereto, the adjustment time of the deflection device according to the invention amounts to approximately 1 ms only. This leads to an overall measuring period of only 3*1 ms (selection of the grating orientation)+9*30 ms (exposure time)+9*1 ms (phase shift via deflection mirrors), thus less than 300 ms.
Preferably, each optical assembly of the first set of optical assemblies comprises one grating. They may show the same grating constants.
In order to generate structured illuminating light of various orientations, the gratings may be arranged in different orientations in reference to each other, which means rotary angles about the optic axis. Grating lines of various gratings are therefore relatively rotated in reference to each other.
Alternatively or additionally, here image field rotators may be provided behind the gratings. They rotate the image of the respectively corresponding grating. This way, all gratings may be arranged in the same orientation, this means showing parallel grating vectors. Here, a grating fastening may be embodied in a simple mechanical fashion, under certain circumstances.
When using image field rotators as the optical assemblies at the optic carrier, in principle a single grating may be sufficient when it is arranged in the radiation path upstream in reference to a deflection device. Any light diffracted by the grating can then be guided to various radiation paths, in which image field rotators are arranged as optical assemblies. With these assemblies differently rotated grating images are generated at the sample plane.
In an embodiment of the optical arrangement according to the invention, at least one of the following optical assemblies is arranged at the optic carrier:
at least one additional set of gratings for generating structured illuminating light, with another set of gratings respectively showing at least three gratings, which show the same grating constant and are particularly arranged in different angles in reference to each other, with the various sets of gratings differing in their grating constants,additional image field rotators for generating differently oriented grating images, with a grating being arranged in the radiation path upstream in reference to the deflection device and it being possible to selectably conduct any light coming from this grating via the deflection device to an image field rotator,a return deflector for providing a wide field illumination, with it being possible to guide a light beam coming from the deflection device with the return deflector back to the deflection device, anda window allowing the light beam to pass through, which comes from the deflection device, with radiation guiding means being provided to guide the light beam allowed to pass to the deflection device.
Conventional optical arrangements generally use only one grating of a single grating constant, because in these embodiments a larger number of gratings leads either to a disproportionately complicated design or to increase dead times during the change of gratings. Contrary thereto, in the optical arrangement according to the invention additional sets of gratings may be present with different grating constants, without this resulting in any significantly increased production expense or dead times. In multi-colored measurements therefore that set of gratings may be selected, which shows the most suitable grating constant for the respective wavelength excited. Various sets of gratings may also be selected by providing several exchangeable optic carriers. An exchange between these optic carriers may be advantageous, for example when the experiment is altered. Due to the fact that here the required exchange period between the optic carriers is of lesser importance, the exchange may occur manually or by a motorized displacement of the optic carriers.
When a reverse mirror or a window is provided at the optic carrier, a more rapid change is possible to an unstructured illumination light. This way, in addition to the examination with structured illumination on a sample image with wide-field illumination, particularly a wide-field epi-illumination can be recorded.
When transmission gratings are used, preferably a window is provided to embody a wide-field illumination. This window may be formed, for example, by a platelet with planar levels, a lens, a group of lenses, or a hole. The radiation guidance means arranged behind the windows for conducting the light beam to the deflection device may, for example, comprise one or more reflectors, lenses, or groups of lenses, as well as a rotational output mirror, which is rotated depending on the adjustment of the deflection device. Preferably, the output mirror is embodied as the back side of a deflection mirror of the deflection device, so that the output mirror and the deflection mirror can be rotated jointly.
In one embodiment of the gratings, the use of a reverse mirror is preferred as a reflection grating for providing a wide-field illumination. It may show a planar or also a curved surface.
Additionally, further sets of gratings may be present at the optic carrier, with respectively one plate with a defined thickness being arranged in front therefore for adjusting the focus. When plates of different thicknesses are provided at various gratings, the depth-related scanning can occur in discrete steps thereby.
It is preferred that the gratings are positioned in a two-dimensional arrangement side-by-side on the object carrier. Using the deflection device, here an impinging light beam can be deflected to an arbitrary grating according to a direction of deflection. When additional optical assemblies are provided at the optic carrier, they are also positioned in the two-dimensional arrangement. In principle, the gratings may also be arranged in a single straight or curved line; however by a two-dimensional arrangement advantageously the differences in the deflection angles to select a grating are smaller for a predetermined number of gratings. With smaller differences in the deflection angles, on the one hand smaller optics may be used, on the other hand the differences are lower in the paths of a light beam from the deflection device to the various gratings. This way it can be largely avoided that only some of the gratings are positioned in an intermediate image level of the light microscope, for example, while the other gratings are located outside the intermediate image level.
In order to further reduce the differences in the paths from the deflection device to the various gratings, additionally, the gratings may be offset in reference to each other in the direction of propagation of a light beam coming from the deflection device. This offset may be selected precisely such that all light paths are of identical length from the deflection device to the gratings. This way, a precise arrangement of all gratings is possible in an intermediate image level.
The deflection device may generally comprise arbitrary, quickly switched means for a variable adjustment of a direction of deflection, for example an acousto-optical device or a rotational or displaceable light-diffracting device.
Rapid switching is achieved with a deflection device having a deflection mirror. This can also be called a scanning mirror and for example, be embodied by a MEMS-scanner. Here, MEMS represents micro-electromechanical system. This scanning mirror may represent the only mobile element to select a grating. Beneficially, here electronic control means are provided and embodied to conduct the light beam to a desired grating or in order for another optical assembly to rotate and/or displace the deflection mirror. In principle, two deflection mirrors may also be provided behind each other, allowing a selection from a larger number of gratings.
Deflection mirrors offer the advantage that the component to be moved is relatively small and thus can be moved rather quickly.
In a variant of the embodiment of the optical arrangement according to the invention the gratings represent reflection gratings for returning an impinging light beam to the deflection mirrors. Thus, the same deflection mirror also deflects the returning light beam. Due to the dual deflection, under certain circumstances, minor imprecisions in positioning of the deflection mirror can be compensated.
When a deflection mirror is used in a Descan-arrangement, which means it conducts a light beam to the optic carrier and back therefrom, the radiation paths of the incoming and outgoing light beam can also coincide on the side of the deflection mirror, facing away from the optic carrier. In particular, in this case, a beam splitter may be provided for conducting a light beam, which originates in a light source of the light microscope, in the direction towards the deflection device and for guiding a light beam, coming from the deflection device, in the direction towards a sample to be examined.
In general, the beam splitter may be of an arbitrary type, and for example, comprise a colored beam splitter or a beam splitter with spatially different transmission and reflection features. However, the beam splitter preferably represents a polarization beam splitter.
In order to allow at the polarization beam splitter to differentiate between light beams, originating in the light source of the light microscope, from light beams coming from the deflection device, preferably means changing polarity are arranged between the polarization beam splitter and the optic carrier. The means changing polarity may, for example, represent a λ/4-plate or a λ/2-plate.
Alight beam originating in the light source of the light microscope shall impinge with a desired polarization the polarization beam splitter and here be forwarded to the deflection device and not directly guided in the direction of the sample. For this purpose, means influencing polarization can be used to adjust a certain direction of polarization between the light source and the polarization beam splitter. The means influencing polarization may show a polarizer or polarization filter and/or a λ/2-plate for changing a direction of polarization of a light beam originating in a light source according to the splitting features of the polarization beam splitter.
In another variant of the embodiment of the optical arrangement, according to the invention, the means influencing polarization can be switched, which are arranged in the radiation path in front of the polarization beam splitter. For example, they may show a switchable polarization filter or a rotational λ/2-plate. This way it can be selected if the light beam is guided from the polarization beam splitter to the deflection device or in the direction of the sample without impinging the deflection device.
When forwarding towards the deflection device the direction of polarization of the light is rotated by means of changing polarization between the polarization beam splitter and the optic carrier before the light returns back to the polarization beam splitter. Accordingly, in this case light is first transmitted at the polarization beam splitter and reflected when coming back, or vice versa, in order to finally reach the sample.
When forwarding occurs at the polarization beam splitter in the direction towards the sample, light is forwarded based on its direction of polarization not in the direction towards the deflection mirror but to a different radiation path. Here, via means changing polarization, the direction of polarization is rotated and the light is guided back to the polarization beam splitter.
For example, a λ/4-plate or a λ/2-plate may be used as a means changing polarization, followed by a reflector. Due to the reflector the λ/4-plate is passed tice, so that the direction of polarization of the light may be rotated by 90° when, coming from the reflector, it once more impinges the polarization beam splitter. Accordingly, in this case the light is first reflected at the polarization beam splitter and transmitted when coming back, or vice versa, in order to reach the sample. This way, the manipulation of the light beam by the grating is avoided. For example, a wide-field illumination can be provided, with any switching towards a structured illumination via the switchable polarization filter or the λ/2-plate being possible before the deflection device within a short period of time and with only minor loss of light. Alternatively, a rapid switching between structured illumination and wide-field illumination is possible in the described embodiments with reverse mirrors at the optic carrier.
When a polarization beam splitter is used, additional means changing polarization are arranged between the polarization beam splitter and the sample to be examined. In principle, here, the polarization changing effect can be compensated of the polarizer, upstream in reference to the beam splitter, and the means changing polarization, arranged between the beam splitter and the optic carrier. Thus, the polarization of a light beam at the time leaving the optical arrangement in the direction towards the sample may coincide with the polarization at the time of entering the optical arrangement. Preferably, these means changing polarization are embodied as rapidly switchable polarization rotators, for example with a rotational λ/2-plate or a switchable liquid crystal section. Electronic control means are provided and embodied such that they adjust the switchable polarization rotator to optimize a modulation contrast in a sample plane depending on the grating orientation of the presently selected grating.
As an alternative to the reflection gratings, here gratings provided at the object carrier may also represent transmission gratings. Instead of a Descan—use of the deflection mirror, here, it may be provided that the deflection device comprises a deflection mirror and an output minor such that with the deflection mirror a light beam originating at the light source of the light microscope, can be deflected to the optic carrier such that the gratings represent transmission gratings to allow a light beam, coming from the deflection mirror, to be deflected in the direction of the output mirror such that with the output mirror a light beam, coming from the optic carrier, can be deflected in the direction of the sample and that electronic control means are provided and implemented for the purpose to jointly rotate and/or displace the output mirror with the deflection mirror.
This way no beam splitter is necessary in order to spatially separate a light beam oriented in the direction of the optic carrier from a light beam coming from said object carrier.
In order to allow a rapid grating selection, the deflection mirrors and the output mirror are adjusted synchronously. This can be realized in a particularly effective fashion when the output mirror and the deflection mirror are fastened at a common fastening for a joint rotation or displacement, which fastening is rotated and/or displaced via the electronic control means. In order for the fastening with the two mirrors to comprise a low torque, the output mirror is formed preferably as the back of the deflection mirror.
Particularly flexible applications are possible in embodiments of an optic arrangement according to the invention, in which the gratings and other optical assemblies are held at the optic carrier in an exchangeable fashion and/or an optic carrier fastening is provided for an exchangeable fastening of the optic carrier. This way, the optic carrier can be equipped with those gratings, which are suitable for the respective light source or light sources used. An exchangeable fastening can occur, for example, by a mechanic plug-in or screwed connection or by a magnetic fixation.
Additionally, variants of the embodiment of the optical arrangement according to the invention are preferred, in which optic focusing means are provided between the deflection device and the optic carrier, in order to guide the respective light beams under identical angles to the respective grating or other optical assemblies of the optic carrier for the various deflection angles of the deflection device. The optic focusing means may particularly represent one or more lenses or groups of lenses. In principle, reflectors or a combination of lenses and reflectors may be used as well. The optic focusing means serve here also for a deflection of a light beam coming from the deflection device. This way, the light beams can extend parallel in reference to each other between the optic focusing means and the optic carrier, independent from the angle of deflection of the deflection device.
Preferably, a light beam is guided by the optic focusing means with its primary direction of propagation being perpendicular in reference to the selected grating. Preferably, light beams are guided with various deflection angles to the same lenses and/or reflectors of the optic focusing mean, however at various sections thereof. Preferably, here a cross-section of the lenses and/or reflectors of the optic focusing means is at least twice as large as the cross-sectional area of the diameter of the light beam at the optic focusing means. Light beams impinging the optic focusing means at various deflection angles can this way be guided and spatially distanced parallel in reference to each other to the optic carrier.
Preferably, the optic focusing means and the deflection device are arranged such that a light beam, reflected by one of the gratings or another optic component, is forwarded by the optic focusing means to the deflection device and further in the direction towards the sample. This way, the optic focusing means also performs a deflecting function when returning a light beam, depending on the angle of deflection of the deflection device.
Another improvement regarding the speed of recording images can be achieved when a certain sample section is displayed successively on various camera sections of a camera. With every selection process of the camera, here, several images of the sample are scanned, which were generated successively on different camera sections.
In this embodiment, at least one camera is provided for recording an image of a section of a sample and a scanning reflector is arranged between the sample plane and the camera. Electronic control means are implemented to display with the scanning reflector the same sample section successively on various sections of the camera.
The scanning reflector may be embodied similar to the deflection mirror in front of the object carrier. Additionally, optic focusing means may also be provided between the scanning reflector and the camera, which similarly to the optic focusing means can be embodied between the deflection mirror and the optic carrier.
Beneficially, the electronic control means are implemented in order to switch the scanning reflector and the deflection mirror at the same points of time. For example, images can be generated on the various camera sections concerning various optic assemblies at the optic carrier.
By the division into several camera sections for each sample image, here, only a portion of all available image pixels of the camera is utilized. Beneficially it may be provided here that a smaller sample section is illuminated than when operating in a manner in which all image pixels of the camera are used to display a single sample image. The illumination of this smaller sample section can be achieved when at least one additional return reflector is provided at the object carrier, with its reflecting section being smaller than in the above-described return reflector. By the fact that the object carrier is arranged in an intermediate image level, only those reflecting sections are displayed in the sample plane and consequently only a smaller sample section is illuminated. This section focused on can also be called region of interest.
In an appropriate fashion, additional gratings and/or windows may be provided at the object carrier, with their widths and/or heights being lower than the other gratings and/or other windows.
The readout time of the camera can also be reduced when only a portion of all image pixels of the camera is being evaluated.
Additionally, a focusing optic or a collimator may be provided for guiding the light beam to the deflection device. Preferably, the electronic control means are arranged for displacing the focusing optic in the direction of propagation of the incident light and/or for adjusting the focal point of the focusing optic. This way an axial displacement of the focal point of the light can be achieved. Alternatively, an axial displacement can also be achieved by the deflection device, the object carrier, as well as all components located there between being displaced jointly in the direction, in which light impinges the deflection device.
The electronic control means may also be embodied in order to displace the deflection device and the focusing optic jointly in the direction of extension of the impinging light beam. For example, a focal point of the light may be laterally displaced in a pupil level of the light microscope. This may occur depending, for example, on the selection of a lens at the light microscope.
By a displacement of the light beam in the lens pupil, for example, a suitable illumination can be adjusted for the internal total reflection—fluorescence microscopy (TIRF). For this method of microscopy, it may additionally be advantageous if one of the optical assemblies is an image field rotator at the optic carrier.
With each grating position a phase shift can occur. This shall be understood as a shift of the illustration of the grating in the sample plane perpendicular with reference to a direction of the grating lines. The shifting can beneficially be smaller than a distance between adjacent grating lines. In order to change a structured illumination, which is generated with one of the gratings, accordingly an actuator or motor may be provided to move the grating. The actuator may represent, for example a Piezo-electric actuator, allowing a position control in two dimensions. Preferably all gratings are arranged in a common fastening and said fastening can be moved via the motor. For example, a single motor or a Piezo-electric actuator is sufficient for moving all gratings.
In order to shift phases of each grating position, alternatively or additionally electronic control means may also be provided to shift or tilt the deflection mirror. For example, light can be conducted successively in at least three different deflection angles to the same grating. Furthermore, a phase shift can be achieved such that an optic-diffractive means are provided between the deflection device and the optic carrier and the electronic control means is provided and implemented for the motorized movement of the optic-diffractive means to change a structured illumination provided with one of the gratings. The optic-diffractive means may represent, for example, a plate or a rocking plate, which is tilted via the electronic control means, or a wedge, which is displaced via the electronic control means.
The displacement or tilting occurs in a direction by which the light beam at the grating is shifted in a direction perpendicular to the direction of the grating lines.
In a preferred variant of the embodiment of the light microscope, according to the invention, the optic carrier is arranged in an intermediate image level or a level of the light microscope conjugated in reference thereto. Thus, a focused display occurs of the selected grating in the sample plane.